WO1999061421A1 - L'oMePUPA-V, NOUVEL INHIBITEUR DU VLA-4 - Google Patents

L'oMePUPA-V, NOUVEL INHIBITEUR DU VLA-4 Download PDF

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Publication number
WO1999061421A1
WO1999061421A1 PCT/US1999/011924 US9911924W WO9961421A1 WO 1999061421 A1 WO1999061421 A1 WO 1999061421A1 US 9911924 W US9911924 W US 9911924W WO 9961421 A1 WO9961421 A1 WO 9961421A1
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Prior art keywords
omepupa
vla
compound
mammal
cell adhesion
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PCT/US1999/011924
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English (en)
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Wen-Cherng Lee
Alan Gill
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Biogen, Inc.
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Priority to EA200001236A priority Critical patent/EA002988B1/ru
Priority to UA2000127603A priority patent/UA65623C2/uk
Application filed by Biogen, Inc. filed Critical Biogen, Inc.
Priority to IL13996799A priority patent/IL139967A/xx
Priority to AT99926019T priority patent/ATE256659T1/de
Priority to MXPA00011774A priority patent/MXPA00011774A/es
Priority to SI9930526T priority patent/SI1082302T1/xx
Priority to EP99926019A priority patent/EP1082302B1/fr
Priority to PL344440A priority patent/PL198189B1/pl
Priority to NZ509199A priority patent/NZ509199A/en
Priority to SK1810-2000A priority patent/SK285280B6/sk
Priority to CA002333656A priority patent/CA2333656C/fr
Priority to JP2000550827A priority patent/JP2002516309A/ja
Priority to HU0102255A priority patent/HUP0102255A3/hu
Priority to EEP200000698A priority patent/EE04639B1/xx
Priority to DK99926019T priority patent/DK1082302T3/da
Priority to AU42192/99A priority patent/AU764108B2/en
Priority to DE69913687T priority patent/DE69913687T2/de
Publication of WO1999061421A1 publication Critical patent/WO1999061421A1/fr
Priority to IS5737A priority patent/IS5737A/is
Priority to US09/724,107 priority patent/US6495525B1/en
Priority to NO20006023A priority patent/NO317990B1/no
Priority to HK01106420A priority patent/HK1035726A1/xx

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
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    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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Definitions

  • a NOVEL VLA-4 LNHmiTOR: oMePUPA-N The present invention relates to novel compounds that are useful for inhibition, alteration, or prevention of cell adhesion and cell adhesion-mediated pathologies. This invention also relates to pharmaceutical formulations comprising these compounds, and methods of using them for inhibition and prevention of cell adhesion and cell adhesion-mediated pathologies.
  • the compounds and pharmaceutical compositions of this invention can be used as therapeutic or prophylactic agents. They are particularly well suited for the treatment of many inflammatory and autoimmune diseases.
  • Cell adhesion is a process by which cells associate with each other, migrate towards a specific target or localize within the extra-cellular matrix.
  • cell adhesion constitutes one of the fundamental mechanisms underlying numerous biological phenomena.
  • cell adhesion is responsible for the adhesion of hematopoietic cells to endothelial cells and the subsequent migration of those hematopoietic cells out of blood vessels and to the site of injury.
  • cell adhesion plays a role in numerous pathologies such as, for example, inflammation and immune reactions in mammals.
  • Investigations into the molecular basis for cell adhesion have revealed that various cell-surface macromolecules — collectively known as cell adhesion molecules or receptors — mediate cell-cell and cell-matrix interactions.
  • Integrins proteins of the superfamily called "integrins” are key mediators in adhesive interactions between hematopoietic cells and their microenvironment (M.E. Hemler, "VLA Proteins in the Integrin Family: Structures, Functions, and Their Role on Leukocytes.” Ann. Rev. Immunol., 8, p. 365 (1990)). Integrins are non-covalent heterodimeric complexes consisting of two subunits called ⁇ and ⁇ .
  • each integrin molecule can be categorized into a subfamily.
  • Integrin ⁇ 4 ⁇ l also known as very late antigen-4 ("VLA-4") or CD49d/CD29, is a leukocyte cell surface receptor that participates in a wide variety of both cell-cell and cell- matrix adhesive interactions (M.E. Hemler, Ann. Rev. Immunol., 8, p. 365 (1990)).
  • VCAM-l vascular cell adhesion molecule- 1
  • FN extracellular matrix protein fibronectin
  • Anti-VLA-4 monoclonal antibodies have been shown to inhibit VLA-4-dependent adhesive interactions both in vitro and in vivo (Ferguson et al. Proc. Natl. Acad. Sci., 88, p. 8072 (1991); Ferguson et al., J. Immunol., 150, p. 1172 (1993)). Results of in vivo experiments suggest that the inhibition of VLA-4- dependent cell adhesion may prevent, inhibit or alter several inflammatory and autoimmune pathologies. (R. L. Lobb et al., "The Pathophysiologic Role of ⁇ 4 Integrins In Vivo", J. Clin. Invest... 94, pp. 1722-28 (1994)). In order to identify the minimum active amino acid sequence necessary to bind
  • VLA-4 Komoriya et al. synthesized a variety of overlapping peptides based on the amino acid sequence of the CS-1 region (the VLA-4 binding domain) of a particular species of fibronectin.
  • CS1 Cell Type-Specific Adhesion Site
  • VLA-5 adhesion to FN has also been described.
  • D.M. Nowlin et al. A Novel Cyclic Pentapeptide Inhibits ⁇ 4 ⁇ l and ⁇ 5Bl Integrin-mediated Cell Adhesion", J. Biol. Chem.. 268(27), pp. 20352-59 (1993); and PCT publication PCT US91/04862).
  • This pentapeptide was based on the tripeptide sequence Arg-Gly-Asp from FN which had been known as a common motif in the recognition site for several extracellular-matrix proteins. Examples of other VLA-4 inhibitors have been reported, for example, in copending United States patent application 08/376,372, specifically incorporated by reference herein.
  • USSN 376,372 describes linear peptidyl compounds containing ⁇ -amino acids which have cell adhesion inhibitory activity.
  • International patent applications WO 94/15958 and WO 92/00995, specifically incorporated by reference, describe cyclic peptide and peptidomimetic compounds with cell adhesion modulating activity.
  • International patent applications WO 93/08823 and WO 92/08464 (specifically incorporated by reference herein) describe guanidinyl-, urea- and thiourea-containing cell adhesion modulating compounds.
  • United States Patent No. 5,260,277 describes guanidinyl cell adhesion modulation compounds, and is also specifically incorporated herein.
  • VLA-4 dependent cell adhesion that have improved pharmacokinetic and pharmacodynamic properties such as oral bioavailability and significant duration of action.
  • Such compounds would provide useful agents for treatment, alteration, prevention or suppression of various pathologies mediated by cell adhesion and VLA-4 binding.
  • the compounds of the present invention are inhibitors of the VLA-4 integrin, thereby blocking the binding of VLA-4 to its various ligands, such as VCAM-1 and regions of fibronectin.
  • VLA-4 integrin a ligand that binds to VLA-4 to its various ligands, such as VCAM-1 and regions of fibronectin.
  • these compounds are useful in inhibiting cell adhesion processes including cell activation, migration, proliferation and differentiation.
  • VLA-4-mediated cell adhesion and pathologies associated with that adhesion such as inflammation and immune reactions, including for example, multiple sclerosis, asthma, allergic rhinitis, allergic conjunctivitis, inflammatory lung diseases, rheumatoid arthritis, septic arthritis, type 1 diabetes, organ transplantation, restenosis, autologous bone marrow transplantation, inflammatory sequelae of viral infections, myocarditis, inflammatory bowel disease including ulcerative colitis and Crohn's disease, certain types of toxic and immune-based nephritis, contact dermal hypersensitivity, psoriasis, tumor metastasis, multiple myeloma, and atherosclerosis.
  • inflammation and immune reactions including for example, multiple sclerosis, asthma, allergic rhinitis, allergic conjunctivitis, inflammatory lung diseases, rheumatoid arthritis, septic arthritis, type 1 diabetes, organ transplantation, restenosis, autologous bone marrow transplantation, inflammatory sequela
  • the compounds of this invention may be used alone or in combination with other therapeutic or prophylactic agents to inhibit, alter, prevent or suppress cell adhesion.
  • This invention also provides pharmaceutical formulations containing these VLA-4-mediated cell adhesion inhibitors and methods of using the compounds and compositions of the invention for inhibition of cell adhesion.
  • BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 reports the airway responsiveness of sheep after treatment with oMePUPA-V. Sheep, naturally sensitive to Ascaris suum, were challenged with an aerosol of Ascaris suum allergen 2 h after aerosol administration of oMePUPA-V at the indicated doses or an equivalent amount of vehicle.
  • Pulmonary mechanics were measured at the indicated times and are reported as the change in specific airways resistance from the pre- study baseline value (left panels). Airways resistance to inhaled carbachol was determined prior to study initiation and at 24 h post-allergen challenge (right panels). Airways responsiveness is reported as the P oo (amount of carbachol required to increase resistance by 400%) ratio by comparison of pre-challenge and post-challenge values
  • Figure 3 Sheep, naturally sensitive to Ascaris suum, were challenged with an aerosol of Ascaris suum allergen 24 h after the fourth daily aerosol administration of oMePUPA-V (0.03 mg) or an equivalent amount of vehicle (ethanoknormal saline, 1:2, upper panel; Tris:normal saline, 1:499, lower panel). Pulmonary mechanics were measured at the indicated times and are reported as the change in specific airways resistance from the pre-study baseline value (left panels). Airways resistance to inhaled carbachol was determined prior to study initiation and at 24 h post-allergen challenge (right panels). Airways responsiveness is reported as the PC 0 o (amount of carbachol required to increase resistance by 400%) ratio by comparison of pre-challenge and post- challenge values.
  • FIG. 4 Balb/c mice, previously sensitized to DNFB, were challenged by application of DNFB to the dorsal surface of the left ear and vehicle to the dorsal surface of the right ear. Twenty-four hours later the thickness of the ears was measured with a micrometer. oMePUPA-V was administered at the indicated doses 4 hours after challenge with DNFB. Positive control (+CTRL) compound was given at a maximally effective enteral dose. Values are means ⁇ standard error of the mean for 8 animals. Upper panel shows absolute ear swelling. Lower panel shows percent inhibition of ear swelling compared to vehicle (VEH) control.
  • VH vehicle
  • FIG. 5 Analysis of competition between OMePUPA-V and a known inhibitor under various conditions of activation.
  • Jurkat cells (1.5 x 10 6 /ml) in TBS plus 2 mM Mn 2+ , 1 mM Ca 2+ plus ImM Mg 2+ , 1 mM Ca 2+ plus 10 mM Mg 2+ , 10 mM Mg 2+ , or 10 mM Mg 2+ plus lO ⁇ g/ml TS2/16 were treated with 5 nM 3 H-known inhibitor alone or 5 nM 3 H-known inhibitor plus 10 nM BIO 1591 for 30 min at room temperature.
  • the cells were then pelleted by centrifugation, resuspended in 100 ⁇ l of TBS plus Mn 2+ , and analyzed by scintillation counting. Counts bound under these conditions measures integrin that is not occupied by the test compound and is therefore free to bind the 3 H-known inhibitor.
  • the present invention provides compounds which are capable of inhibiting VLA-4 mediated cell adhesion by inhibiting the binding of ligands to that receptor.
  • the preferred compound is (R)-N-[[4-[[(2-methylphenylamino) carbonyl]amino]phenyl]acetyl]-L-prolyl- 3-methyl)- ⁇ -Alanine, referred to herein as "oMePUPA-V", represented by the following formula I:
  • oMePUPA-V oMePUPA-V
  • the invention is also intended to encompass pharmaceutically acceptable derivatives, salts, and esters of oMePUPA-V.
  • Compounds of Formula I contain one or more asymmetric centers and thus can occur as racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention is meant to comprehend all such isomeric forms of the compound of Formula I.
  • the claimed invention is also intended to encompass pharmaceutically acceptable salts of Formula I.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N- ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropyulamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
  • basic ion exchange resins
  • salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.
  • acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.
  • Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.
  • the claimed invention encompasses prodrugs, specifically, ester prodrugs wherein the carboxyl group of:
  • (R)-N-[[4-[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl]-L-prolyl-3-methyl)- ⁇ - Alanine is esterified with any of the alcohols.
  • Preferred alcohols are methanol, ethanol, propanol, butanol, or straight or branched chain alkyl C 1 - 10 alcohols.
  • VLA-4 antagonizes the actions of VLA-4
  • these antagonists will inhibit cell adhesion processes including cell activation, migration, proliferation and differentiation.
  • diseases and disorders include, for example, asthma, multiple sclerosis, allergic rhinitis, allergic conjunctivitis, inflammatory lung diseases, rheumatoid arthritis, multiple myeloma, septic arthritis, type I diabetes, organ transplant rejection, inflammatory bowel disease, and others.
  • Compounds of this invention may be synthesized using any conventional technique, several of which are exemplified herein.
  • these compounds are chemically synthesized from readily available starting materials, such as ⁇ -amino acids and their functional equivalents. Modular and convergent methods for the synthesis of these compounds are also preferred. In a convergent approach, for example, large sections of the final product are brought together in the last stages of the synthesis, rather than by incremental addition of small pieces to a growing molecular chain.
  • the compounds of this invention may also be modified by appending appropriate functionalities to enhance selective biological properties.
  • Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion. Examples of these modifications include, but are not limited to, esterification with polyethylene glycols, derivatization with pivolates or fatty acid substituents, conversion to carbamates, hydroxylation of aromatic rings, and heteroatom-substitution in aromatic rings.
  • the term "patient” refers to mammals, including humans.
  • the term “cell” refers to any cell, preferably mammalian cells, including human cells.
  • the activities and VLA-4 specificities of the compounds according to this invention may be determined using in vitro and in vivo assays.
  • the cell adhesion inhibitory activity of these compounds may be measured by determining the concentration of inhibitor required to block the binding of
  • VLA-4-expressing cells to fibronectin- or CS 1 -coated plates.
  • microtiter wells are coated with either fibronectin (containing the CS-1 sequence) or CS-1.
  • CS-1 CS-1
  • a carrier protein such as bovine serum albumin
  • test compound may be added first and allowed to incubate with the coated wells prior to the addition of the cells.
  • the cells are allowed to incubate in the wells for at least 30 minutes.
  • the wells are emptied and washed. Inhibition of binding is measured by quantitating the fluorescence or radioactivity bound to the plate for each of the various concentrations of test compound, as well as for controls containing no test compound.
  • VLA-4-expressing cells that may be utilized in this assay include Ramos cells,
  • VCAM-IgG fusion protein containing the first two immunoglobulin domains of VCAM (D1D2) attached above the hinge region of an IgGl molecule (“VCAM 2D-IgG”), is conjugated to a marker enzyme, such as alkaline phosphatase ("AP").
  • AP alkaline phosphatase
  • VCAM-IgG fusion The synthesis of this VCAM-IgG fusion is described in PCT publication WO 90/13300, the disclosure of which is herein incorporated by reference.
  • conjugation of that fusion to a marker enzyme is achieved by cross-linking methods well-known in the art.
  • VCAM-IgG enzyme conjugate is then placed in the wells of a multi-well filtration plate, such as that contained in the Millipore Multiscreen Assay System
  • VCAM-IgG enzyme conjugate is then added to the wells followed by addition of VLA-4-expressing cells.
  • the cells, compound and VCAM-IgG enzyme conjugate are mixed together and allowed to incubate at room temperature. Following incubation, the wells are vacuum drained, leaving behind the cells and any bound VCAM. Quantitation of bound VCAM is determined by adding an appropriate colorimetric substrate for the enzyme conjugated to VCAM-IgG and determining the amount of reaction product. Decreased reaction product indicates increased binding inhibitory activity.
  • assays for other major groups of integrins i.e., 62 and 63, as well as other 61 integrins, such as VLA-5, VLA-6 and ⁇ 467 are performed.
  • these assays may be similar to the adhesion inhibition and direct binding assays described above, substituting the appropriate integrin-expressing cell and corresponding ligand.
  • polymo ⁇ honuclear cells PMNs express 62 integrins on their surface and bind to ICAM.
  • B3 integrins are involved in platelet aggregation and inhibition may be measured in a standard platelet aggregation assay.
  • VLA-5 binds specifically to Arg-Gly-Asp sequences, while VLA-6 binds to laminin.
  • ⁇ 467 is a recently discovered homologue of VLA-4, which also binds fibronectin and VCAM. Specificity with respect to ⁇ 467 is determined in a binding assay that utilizes the above-described VCAM-IgG-enzyme marker conjugate and a cell line that expresses ⁇ 4B7, but not VLA-4, such as RPMI-8866 or JY cells.
  • VLA-4-specific inhibitors Once VLA-4-specific inhibitors are identified, they may be further characterized in in vivo assays.
  • One such assay tests the inhibition of contact hypersensitivity in an animal, such as described by P.L. Chisholm et al., "Monoclonal Antibodies to the Integrin ⁇ -4
  • one ear of the animal is exposed to the chemical irritant, while the other ear is treated with a non-irritant control solution.
  • the animals are given various doses of the VLA-4 inhibitor by subcutaneous injection.
  • In vivo inhibition of cell adhesion-associated inflammation is assessed by measuring the ear swelling response of the animal in the treated versus untreated ear. Swelling is measured using calipers or other suitable instrument to measure ear thickness. In this manner, one may identify those inhibitors of this invention which are best suited for inhibiting inflammation.
  • sheep asthma assay Another in vivo assay that may be employed to test the inhibitors of this invention is the sheep asthma assay. This assay is performed essentially as described in W. M. Abraham et al., " ⁇ 4-Integrins Mediate Antigen-induced Late Bronchial Responses and Prolonged Airway Hyperresponsiveness in Sheep", J. Clin. Invest., 93, pp. 776-87 (1994), the disclosure of which is herein incorporated by reference. This assay measures inhibition of Ascaris antigen-induced late phase airway responses and airway hyperresponsiveness in allergic sheep. The compounds of this invention may also be tested in a platelet aggregation assay.
  • VLA-4 inhibitors of the invention have shown surprisingly favorable activity and selectivity. Generally, these compounds are selective for VLA-4 (> 1000-fold vesus ⁇ 4 ⁇ 7 and ⁇ 5 ⁇ l), negative in routine PanLabs and non-GLP Ames assays, clean in standard ancillary pharmacology tests and effective in the sheep model following once-a- day dosing at a predicted use level in man of 1 mg/day or less.
  • the claimed compounds have su ⁇ risingly superior potency as compared to structurally related VLA-4 inhibitors.
  • oMePUPA-V is 30 to 100-fold more potent than previous compounds, with dose levels in the range of the best marketed inhaled steroids.
  • oMePUPA-V, as well as the penultimate synthetic intermediate, is highly crystalline. (See Figure 1, Table 1)
  • oMePUPA-V has an improved metabolic profile as compared to known VLA-4 inhibitor compounds.
  • the claimed compounds were rapidly converted to a less active metabolite, which was the predominant product recovered from bronchoalveolar lavage fluid (BALF) and was also the predominant product observed in the systemic circulation where it exhibited a longer plasma half-life than the parent compound. While rapid metabolic conversion to less active compounds is a useful strategy to achieve reduced systemic exposure, compounds which are metabolized to by-products with little or no intrinsic activity present fewer complexities in development and are preferred from a backup perspective.
  • BALF bronchoalveolar lavage fluid
  • compositions that may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • compositions of this invention comprise any of the compounds of the present invention, or pharmaceutically acceptable derivatives thereof, together with any pharmaceutically acceptable carrier.
  • carrier includes acceptable adjuvants and vehicles.
  • Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as do natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long- chain alcohol diluent or dispersant.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried corn starch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • compositions of this invention may be administered in the form of suppositories for rectal administration.
  • suppositories for rectal administration.
  • suppositories can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter, beeswax and polyethylene glycols.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including, for example, diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically- transdermal patches may also be used.
  • the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride.
  • the pharmaceutical compositions may be formulated in an ointment such as petrolatum.
  • compositions of this invention may also be administered by nasal aerosol or inhalation through the use of a nebulizer, a dry powder inhaler or a metered dose inhaler.
  • a nebulizer a dry powder inhaler or a metered dose inhaler.
  • Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, abso ⁇ tion promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • the compositions of the invention may include any pharmaceutically acceptable carriers, such as, for example, lactose for dry powder formulations.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, and the particular mode of administration. It should be understood, however, that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of active ingredient may also depend upon the therapeutic or prophylactic agent, if any, with which the ingredient is co-administered.
  • the dosage and dose rate of the compounds of this invention effective to prevent, suppress or inhibit cell adhesion will depend on a variety of factors, such as the nature of the inhibitor, the size of the patient, the goal of the treatment, the nature of the pathology to be treated, the specific pharmaceutical composition used, and the judgment of the treating physician. Dosage levels of between about 0.001 and about 100 mg/kg body weight per day, preferably between about 0.01 to about 50 mg/kg and more preferably about
  • a suitable dosage range is from about 0.001 mg to about 25 mg/kg, more preferably, about 0.01 mg to about 1 mg/kg.
  • compositions containing a compound of this invention may also comprise an additional agent selected from the group consisting of corticosteroids, bronchodilators, antiasthmatics (mast cell stabilizers), antiinflammatories, antirheumatics, immunosuppressants, antimetabolites, immunonodulators, antipsoriatics and antidiabetics.
  • additional agent selected from the group consisting of corticosteroids, bronchodilators, antiasthmatics (mast cell stabilizers), antiinflammatories, antirheumatics, immunosuppressants, antimetabolites, immunonodulators, antipsoriatics and antidiabetics.
  • Specific compounds within each of these classes may be selected from any of those listed under the appropriate group headings in "Comprehensive Medicinal Chemistry", Pergamon Press, Oxford, England, pp. 970-986 (1990), the disclosure of which is herein inco ⁇ orated by reference.
  • Also included within this group are compounds such as theophylline, sulfasalazine and aminosalicylates (antiinflammatories); cyclosporin, FK-506, and rapamycin (immunosuppressants); cyclophosphamide and methotrexate
  • the compounds of the invention may be administered in conjunction with additional cell adhesion inhibitors.
  • additional agents in combination with the claimed VLA-4 inhibitor
  • the active ingredients may be formulated together, or, alternatively may be administered in combination.
  • Administration of one or more active agents in combination with the VLA-4 inhibitors of the claimed invention may be substantially simultaneous, or sequential.
  • the invention provides methods for preventing, inhibiting or suppressing cell adhesion-associated inflammation and cell adhesion- associated immune or autoimmune responses in a patient.
  • VLA-4-associated cell adhesion plays a central role in a variety of inflammation, immune and autoimmune diseases.
  • inhibition of cell adhesion by the compounds of this invention may be utilized in methods of treating or preventing inflammatory, immune and autoimmune diseases.
  • the diseases to be treated with the methods of this invention are selected from asthma, arthritis, psoriasis, transplantation rejection, multiple sclerosis, diabetes and inflammatory bowel disease.
  • These methods may employ the compounds of this invention in a monotherapy or in combination with an anti-inflammatory or immunosuppressive agent.
  • Such combination therapies include administration of the agents in a single dosage form or in multiple dosage forms administered at the same time or at different times.
  • Mass Spectra were run on a Fisons VG Platform LC-MS-DS Mass Spectrometer System with a Hewlett Packard Model 1500 AutoSampler and the data processed using a Fisons VG MassLynx Mass Spectrometer Workstation.
  • the HRMS work was run at M- Scan (PA) using Fast Atom Bombardment on a VG Analytical ZAB 2SE high field mass spectrometer with reference to SOP# MS-002, MS-006, MS-012 and MS-023.
  • a cesium ion gun was used to generate ions for the acquired mass spectra which were recorded using a PDP-11-250J data system.
  • IR spectra were performed on a Perkin Elmer 1600 Series FTE .
  • Analytical HPLC chromatography was performed as follows: 1. Chromatograms using Program 1 (Equilibrate @ 20% B, inject sample, 20% B (1 min.), 20% - 70% B (24 min.), 70% - 100% B (17 min.) were obtained using a Perkin Elmer Series 200 HPLC autosampler system with a Perkin Elmer 785A UV detector (set at 214 nm) and an Applied Biosystems 783 A UV detector (set at 254 nm) with a PE Nelson 1020 integrator. Only the area percent values were reported. 2.
  • MPUPA-OH material manufactured by Ricerca Inc.
  • Celgene Co ⁇ . mp 249.4 - 249.8°C; IR (KBr) 3515, 3383, 2989, 2945, 2880, 1821, 1788, 1744, 1701, 1476, 1454, 1421, 1394,
  • the reaction mixture was partitioned between EtOAc (1.5 L) and H 2 O (250 mL) and the organic layer was washed with 10% citric acid (3 x 250 mL), H 2 O (250 mL), saturated sodium bicarbonate (250 mL), H 2 O (250 mL), and brine (3 x 250 mL), dried over Na 2 SO 4 and concentrated first on the rotavap (40 °C; -80 mmHg) and then under high- vacuum (room temperature, 16 h; 0.2 mmHg) to provide intermediate 5 as a viscous oil (88.1 g) that contained residual EtOAc and CH 2 C1 2 (by NMR) and exhibited purity >98% (HPLC).
  • the reaction mixture was allowed to stir at room temperature (2 h) and it was then concentrated first on the rotavap (45°C, -80 mmHg) and then under high-vacuum overnight (room temperature, 14 h, -0.2 mmHg) to provide an extremely viscous material which was crystallized from CH 2 Cl 2 /Et 2 O (600 mL/700mL) to provide 64.0 g (92 % overall yield for two steps) of the HCI salt 6 as a white solid (HPLC purity 99.6 %).
  • Pleural pressure was estimated using an esophageal balloon catheter (filled with 1 mL of air), which was positioned 5 to 10 cm from the gastroesophageal - junction. In this position, the end expiratory pleural pressure ranged between -2 to -5- cmH 2 O. Once the balloon was placed, it was secured so that it remained in the same position for the duration of the experiment. Lateral pressure in the trachea was measured with a sidehole catheter (inner diameter, 2.5 mm) advanced through and positioned distal to the tip of the endotracheal tube.
  • the tracheal and pleural pressure catheters were connected to a differential pressure transducer (MP45, Validyne, Northridge, CA) for the measurement of transpulmonary pressure which was defined as the difference between tracheal and pleural pressure.
  • Airflow was measured by connecting the proximal end of the endotracheal tube to a pneumotachograph (Fleisch, Dyna Sciences, Inc., Blue Bell, PA).
  • the signals of transpulmonary pressure and flow were recorded on a multichannel physiological recorder which was linked to a 80-386 DOS Personal Computer for on-line calculation of mean pulmonary flow resistance (R L ) by dividing the change in transpulmonary pressure by the change in flow at mid-tidal volume (obtained by digital integration).
  • the nebulizer was connected to a dosimeter system, consisting of a solenoid valve and a source of compressed air (20 psi). The output of the nebulizer was directed into a plastic T-piece, one end of which was connected to the inspiratory port of a piston respirator (Harvard Apparatus, S. ⁇ atick, MA). The solenoid valve was activated for one second at the beginning of the inspiratory cycle of the respirator.
  • Aerosols were delivered at a tidal volume of 500 mL and a rate of 20 breaths per minute.
  • cumulative concentration response curves to carbachol were performed by measuring SR L immediately after inhalation of buffer and after each consecutive administration of 10 breaths of increasing concentrations of carbachol (0.25, 0.5, 1.0, 2.0 and 4.0% w/v in buffered saline). The provocation test was discontinued when SR L increased over 400% from the post-saline value or after the highest carbachol concentration had been administered.
  • Bronchial responsiveness was assessed by determining the cumulative carbachol concentration (in breath units) that increased SR L by 400% over the post-saline value (PC 40 o) by inte ⁇ olation from the dose response curve.
  • One breath unit (BU) was defined as one breath of a 1 % w/v carbachol nebulized solution.
  • oMePUPA-V Doses of oMePUPA-V were dissolved in either ethanol: normal saline 1:2, ethanol:200 mM sodium phosphate 1:5 or Tris buffer. When using Tris, any required dilution was performed using normal saline. Doses were prepared in 3-5 mL total volume. In all studies, baseline airway responsiveness (i.e., PG»oo) was determined three to four days before initiating a study. In single-dose pre-treatment studies, SR L was measured and animals were treated with the compound or with vehicle. SR L was remeasured 2 hours after treatment (just before challenge) and then the animals were challenged with allergen.
  • PG airway responsiveness
  • SR L was measured before and after the last dose of compound or vehicle treatment. In all studies, SR L was remeasured immediately after allergen challenge, hourly from 1-6 hours after challenge, and half-hourly from 6.5-8 hours after allergen challenge. Post-challenge determinations of airway responsiveness (PC 40 o) were made 24 hours after allergen challenge.
  • SR L Change in SR L was calculated for each sheep as the difference from pre-challenge baseline SR L - Post- challenge changes in SR L were characterized by an early airway response (EAR) which evolved over the approximately 0-4-hour period. This was followed by a late airway response (LAR) that evolved over the approximately 4-8-hour period after allergen challenge. Areas under the EAR and LAR curves were computed for each animal using the trapezoidal rule. Significant reductions in area under the EAR or LAR curves compared to placebo control were taken to be therapeutic effects on allergen-induced changes in SR L .
  • EAR early airway response
  • LAR late airway response
  • PC 40 o Airway responsiveness to carbachol assessed before, and at 24 h after allergen challenge, was expressed as a PC 40 o ratio (post/prechallenge PC 4 oo values) for each sheep. A significant increase in the PC 4 oo ratio compared to placebo control was taken to be a therapeutic effect. Comparisons to placebo control were made using one-way analysis of variance followed by Dunnett's test (1 -tailed) for multiple comparison to a control. Comparisons that resulted in p ⁇ 0.05 were taken to be statistically significant.
  • Figure 1 shows aerosolized oMePUPA-V's inhibitory dose-response in Ascaris swwm-sensitive sheep challenged 2 h after dosing.
  • Left panels display change in specific lung resistance SR L , cm H 2 O/sec.
  • Right panels display airway responsiveness to inhaled carbachol (PQoo ratio, pre/post-challenge) determined at 24 h after challenge.
  • oMePUPA- V at doses of 0.01 and a 0.03mg did not inhibit early or late airway response or alter hyperresponsiveness to carbachol at 24 h after allergen challenge.
  • Doses of 0.1, 1 and 3 mg inhibited the early airway response and maximally inhibited the late airway response.
  • Figure 3 illustrates that a 0.03 mg dose of oMePUPA-V, which was shown to be ineffective when used as a single dose acute pretreatment, was nevertheless protective if given once daily for 4 days, when antigen challenge was given 24 h after the last dose.
  • the upper and lower left hand panels show that this effect was seen using two different formulations. Hyperresponsiveness to carbachol after a further 24 h was also maximally inhibited as shown in the upper and lower right hand panels of Figure 3.
  • the protective effect of oMePUPA-V was significant against EAR and LAR and against hyperresponsiveness to carbachol and the quantitative analysis is shown in Table 3.
  • mice Specific pathogen-free female Balb/c mice, aged 8-10 weeks, from Jackson Labs were used for all experiments. The animals were fed food and water ad libitum. Sheep red blood cells (sRBC) in Alsever's solution from the same sheep were obtained weekly from
  • the sRBC were pelleted by centrifugation at lOOOg for 10 minutes at 4°C and any visible buffy coat removed. The cells were then washed in saline. The cell pellet was resuspended in saline and counted using a hemocytometer. The cells were diluted in phosphate buffered saline (PBS) to
  • mice 8 7 2x10 sRBC per mL.
  • mice were sensitized by a s.c. injection of 2x10 sRBC in
  • oMePUPA-V (Lot# 2770-029) was formulated in a vehicle of 60% PEG 400 in 0.02M TRIS to a stock concentration of 5 mg/mL. Appropriate dilutions were prepared in the PEG/TRIS vehicle and administered enterally in a volume of 100 ⁇ L.
  • the anti- VLA-4 antibody (PS/2) was diluted in saline at a concentration of 4.3 mg/kg and administered intraperitoneally in 100 ⁇ L. All treatments were administered immediately following challenge with sRBC.
  • mice Twenty gram female virus-free Balb/c mice (Jackson Laboratories, Bar Harbor, ME) housed four to a cage in microisolator cages in Biogen's virus-free animal facility and receiving ad libitum mouse chow and tap water were used for all studies. Mice were anesthetized with ketamine:xylazine (90:10 mg/kg, i.p.). A 3 cm 2 patch of abdominal skin, xiphoid to pubis was exposed by closely shaving the fur and the skin was scrubbed with 70% ethanol. A 25 ⁇ L volume of 0.5% DNFB in 4: 1 v/v acetone:olive oil vehicle is uniformly applied to the bare abdominal skin.
  • mice were again sensitized with 25 ⁇ L of 0.5% DNFB in vehicle at the same abdominal skin location, again followed by light scratching with the pipette tip. The second sensitization was performed while restraining the unanesthetized mouse.
  • a subirritant dose of the sensitizer (0.2% DNFB in 4:1 v/v acetone:olive oil vehicle) was used to challenge the immune response.
  • mice were anesthetized with 90: 10 mg/kg ketamine:xylazine, i.p. and 10 ⁇ L of 0.2% DNFB was applied to the dorsal surface of the left ear.
  • the right ear received a similar application of the 4: 1 v/v acetone:olive oil vehicle.
  • a biphasic ear swelling response evolved, as shown in Figure 4.
  • mice were again anesthetized with ketamine:xylazine and the ear thickness of both ears measured with an engineer's micrometer to an accuracy of 10 inches.
  • Figure 4 compares ear swelling responses measured 24 hours after DNFB challenge in mice that received vehicle (DMSO, PBS), positive control compound (given at 0.03 ⁇ g/kg), or 0.03 or 0.3 mg/kg oMePUPA-V, dosed enterally 4 hours after DNFB challenge
  • DNFB challenge can significantly inhibit the ear swelling response in a model of mouse contact hypersensitivity.
  • VCAM-Ig was constructed and purified as published (Jakubowski, A. et al. Cell Adhesion and Communication 3:131-142, 1995). Conjugation to calf intestinal alkaline phosphatase, for pu ⁇ oses of cleaving a chromogenic substrate, was performed as published (Lobb, R.R. et al. Cell Adhesion and Communication 3:385-397, 1995).
  • VLA-4 Binding to VLA-4 was assessed on the human T cell line, Jurkat ( ⁇ 4 ⁇ l). VCAM-Ig- AP and oMePUPA-V competed for binding to ⁇ 4 ⁇ l on the surface of these cells in the presence of 1 mM Mn +2
  • oMePUPA-V competes with VCAM-Ig-
  • VLA-4 Receptor affinity of oMePUPA-V as measured using VCAM-Ig Alkaline Phosphatase Conjugate in the Purified VLA-4 Protein/Protein Assay
  • VLA-4 was purified from a detergent extract of a high expressing subclone of ⁇ 4- transfected K562 cells by antibody affinity chromatography and immobilized on microtiter wells to establish a protein/protein competitive binding assay.
  • VCAM-Ig- AP was bound to the purified VLA-4-coated plate in the absence or presence of oMePUPA-V (Lot #2) and 1 mM MnCl 2 . Plates were read at 405 nm and the data were analyzed using SoftMax v. 2.32 software.
  • SMCC is a heterobifunctional crosslinker which reacts with free amino groups on BSA and the sole cysteine of the synthetic peptide
  • CS 1/BS A ratio of 10: 1 Wells were coated overnight with 100 ⁇ L of conjugate diluted to a final concentration of 1 ug/ml. The next day the wells were blocked with BSA in PBS for one hour and then washed three times.
  • the human T cell line, Jurkat was labeled with 2 ⁇ M BCECF-AM, a fluorescent dye (2', T , bis-(2-carboxyethyl)-5 and -6) carboxy fluorescein acetoxymethyl ester (Molecular Probes Inc., Eugene, Oregon; catalog #B-1150) that is internalized and deesterified thus trapping the dye within live cells.
  • Jurkat cells (1 x 10 5 cells/well) in buffer containing 1 mM Mn +2 were added to the coated plates in the presence of three-fold serial dilutions of inhibitor. Each concentration was assayed in duplicate. After 30 minutes at room temperature, the plates were inverted and washed three times or until no cells were adherent to control wells coated with BSA alone.
  • CS 1 -adherent cells were quantitated in a Cytofluor fluorescent platereader using an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
  • Cells adhered to CS 1/BS A in the absence of compound served as the 0% inhibition control whereas cells adhering to BSA alone served as the 100% inhibition control.
  • IC50's were calculated using Deltagraph software, version 5.
  • oMePUPA-V Adhesion of labeled Jurkat cells in the presence of Mn +2 was blocked completely by EDTA and the neutralizing anti- ⁇ 4 ⁇ l mAb, HP 1/2, indicating that binding was specific.
  • Table 4 gives the activity of oMePUPA-V in the CS 1/BS A adhesion assay, as well as the binding assay results.
  • oMePUPA-V is a potent VLA-4 antagonist in buffers containing Mn +2 . It is 80- fold more potent when assayed in the presence of Mn +2 on isolated VLA-4 than on Jurkat cells in the binding assay.
  • oMePUPA-V is a functional antagonist as revealed by its ability to dose-dependently and completely block adhesion of Jurkat to CSl. The absolute values in the adhesion assay are greater than those observed in the binding assays. This may be due to the multivalent nature of adhesion. In all assay formats, inhibition of binding by EDTA and HP 1/2
  • Binding to ⁇ 4 ⁇ 7 was assessed on JY cells in the presence of Mn +2 .
  • VCAM-Ig and oMePUPA-V compete for binding to ⁇ 4 ⁇ 7 on JY cells (See section 4.1.1 for assay protocol).
  • oMePUPA-V was tested for its ability to block JY ( ⁇ 4 ⁇ 7) cell binding to CS1/BSA conjugate oMePUPA-V does not block ⁇ 4 ⁇ 7 binding to VCAM-Ig or CS1/BSA.
  • the anti- ⁇ 7 Mab, Fib27 (Pharmingen) inhibited these interactions completely indicating that binding was ⁇ 4 ⁇ 7 specific. Therefore oMePUPA-V is a specific inhibitor for VLA-4. Results are tabulated in Table 5.
  • Activity against gpllbllla was assessed by means of standard platelet aggregometry using platelet rich plasma.
  • ADP was used to initiate aggregation in the presence of plasma where Ca +2 and Mg +2 are the major divalent cations.
  • GRGDSP @ 100 ug/mL was used as a positive control.
  • oMePUPA-V was tested at three doses 1, 10 and 100 ⁇ M. It did not inhibit platelet aggregation as induced by ADP, at any dose. Results are listed in Table 5.
  • oMePUPA-V is highly (>10,000 fold) specific for VLA-4. It has no measurable activity (>100 ⁇ M) against the related integrins, ⁇ 4 ⁇ 7 and VLA-5 or against the ⁇ 3 integrin, gpHbllla.
  • the cells were washed twice with Mg +2 -TBS and resuspended in a 1: 200 dilution of a FTTC conjugated-goat anti-rat IgG in Mg +2 -TBS and incubated for 30 min at 4°C.
  • the cells were washed twice and resuspended in Mg +2 -TBS.
  • Mean fluorescence intensity (MFI) was determined by FACS analysis (Becton Dickinson FACScan). Results are expressed as MFI. Data were analyzed by Microsoft Excel v5.0 and Deltagraph v4.0 software.
  • Fig. 5 shows that oMePUPA-V induced the exposure of the LIBS epitope as compared to 2 mM Mg +2 buffer (Panel B).
  • the induction was concentration dependent and similar in magnitude to the induction observed with 1 mM Mn +2 (Panel A).
  • the ED50 of the response was -20 nM.
  • the Multi-species Receptor Screen a Receptor affinity of oMePUPA-V as measured in VCAM-Ig Direct Binding Assay using VCAM-Ig Alkaline Phosphatase Conjugate and peripheral blood lymphocytes or spleen cells from various species.
  • PBLs were isolated from peripheral blood of humans, sheep and dogs using methods described for sheep PBL (Abraham, W. M. et al. J. Clin. Invest. 93:776-787,
  • VCAM-Ig- AP Competitive Binding Assay was used to compare the binding of oMePUPA-V to these different cell types.
  • IC50's obtained for oMePUPA-V on peripheral blood lymphocytes or spleen cells from various species in the presence of Mn +2 are shown in Table 6.
  • Mn +2 oMePUPA-V inhibits with a similar IC 50 the binding of VCAM-Ig to lymphocytes obtained from humans, rats, dogs, sheep, and mice.
  • species specificity This is consistent with the high degree of sequence conservation observed among species for VLA-4 and its natural ligands, CS-1 and VCAM.
  • Jurkat cells were maintained in RPMI-1640 medium plus 10% fetal bovine serum at 37°C in a tissue culture incubator. For binding studies, the cells were pelleted by centrifugation, washed two times with TBS (50 mM Tris HCI, 150 mM NaCl, 0.1% bovine serum albumin, 2 mM glucose, 10 mM HEPES pH 7.4), suspended at approximately 2 x 10 6 cells/ml in TBS, and counted using a Neubauer hemocytometer. The cells were further diluted to 1.5 x 10 6 /ml in the buffers indicated and subjected to the specific treatments defined for each experiment.
  • TBS 50 mM Tris HCI, 150 mM NaCl, 0.1% bovine serum albumin, 2 mM glucose, 10 mM HEPES pH 7.4
  • the cells were then pelleted by centrifugation, resuspended in 100 ⁇ l of assay buffer, and transferred to a scintillation vial containing 2.9 ml of ScintiVerse II (Fisher Scientific). Cell-associated radioactivity was quantified by scintillation counting. Counts bound under these conditions measures integrin that is not occupied by the test compound and is therefore free to bind the 3 H-known inhibitor. All studies were performed in siliconized 1.5 ml eppendorf tubes with a standard 1 ml sample volume. Non-specific binding of the 3 H-known inhibitor to cells (background) was defined by measuring the inhibitor bound in the absence of metal ion. Specific counts bound were calculated by subtracting non-specific counts from total counts bound.
  • oMePUPA-V A series of competition studies were performed to verify that oMePUPA-V and the known inhibitor compete for the same site on VLA-4.
  • the 3 H-known inhibitor was mixed with an equimolar amount of oMePUPA-V, a 10-fold excess, and a 100-fold excess, incubated with Jurkat cells and the ability of the cold inhibitor to compete for binding of the known inhibitor assessed.
  • oMePUPA-V treatment produced a dose-dependent inhibition of 3 H-known inhibitor binding.
  • the concentration of oMePUPA-V that was needed to compete 3 H-known inhibitor binding was 10-fold greater than was needed when cold was used as a competitor, consistent with its lower affinity for Mn +2 -activated VLA-4.
  • Mn +2 -activated Jurkat cells were treated with 3 H-known inhibitor in order to first occupy VLA-4 with the radioactive probe and then excess cold oMePUPA-V was added. Subsequent treatments with excess cold oMePUPA-V or known inhibitor were indistinguishable in their ability to displace the radioactive probe.
  • Mn +2 -activated Jurkat cells were treated with saturating amounts of oMePUPA-V, and the rate at which the oMePUPA-V dissociated was measured.
  • oMePUPA-V is rapidly released from the oMePUPA- V- VLA-4 with a t 1 2 of less than 10 min.
  • t 1 2 of less than 10 min.
  • the large difference in t 1 2 for oMePUPA-V and the known inhibitor suggests that the lower affinity of oMePUPA-V for VLA-4 is a result of its faster off rate.
  • Dissociation data reveals that binding of oMePUPA-V to VLA-4 is highly dependent on the activation state of VLA-4 and that it exhibits the same selectivity for activation seen with the known inhibitor.
  • the ty 2 of oMePUPA-V dissociation from Mg +2 -activated VLA-4 was less than 10 min the shortest time point that can be assessed in the competition format.
  • TS2/16 the t 1 2 was prolonged (20 min). All of the possible activation states have not been assessed in detail, however a simple screen was devised that can rapidly highlight differences.
  • oMePUPA-V 10 nM
  • 5 nM 3 H-known inhibitor binding was performed under these conditions at various states of activation. If oMePUPA-V had an abnormally high or low affinity for VLA-4 one would detect this by the difference in the amount of 3 H-known inhibitor. The differences in percentage of 3 H-known inhibitor bound under different activation conditions approximate what would be predicted based on the known properties of the inhibitor.
  • ACE proteases were human endothelial cells (HUVEC).
  • 3 H-HGG added to HUVEC, was converted to 3 H-hippuric acid and glycylglycine by ACE.
  • Captopril a potent ACE inhibitor, blocked the conversion with an IQo of 990 pM, while, oMePUPA-V at 10 ⁇ M, did not.
  • oMePUPA-V is a white to off-white crystalline powder. It is soluble in DMSO and has an aqueous solubility of 0.120 mg/mL.
  • the thermal behavior of oMePUPA-V studied by DSC, TGA and hot stage microscopy indicates that the material melts at approximately 160°C. At approximately 136°C the DSC and TGA analyses suggest that oMePUPA-V loses a volatile impurity which maybe consistent with the dehydration of a monohydrate.
  • Nebulization Formulation The manufacturing directions for 100 mL of a 5 mg/mL oMePUPA-V nebulizationformulation are listed below. Prepare 200 mL stock buffer solution as follows:
  • Control oMePUPA-V, heated in boiling water for 20 min.

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Abstract

L'invention porte sur l'oMePUVA-V, (R)-N-[[4-[[(2-méthylphénylamino)carbonyl]amino]phényl]acétyl]-L-prolyl-3-méthyl)-β-Alanine, inhibiteur de l'adhésion cellulaire, sur des compositions pharmaceutiques, et sur des procédés de traitement de pathologies induites par l'adhésion cellulaire.
PCT/US1999/011924 1998-05-28 1999-05-28 L'oMePUPA-V, NOUVEL INHIBITEUR DU VLA-4 WO1999061421A1 (fr)

Priority Applications (21)

Application Number Priority Date Filing Date Title
AU42192/99A AU764108B2 (en) 1998-05-28 1999-05-28 A novel VLA-4 inhibitor: oMePUPA-V
SK1810-2000A SK285280B6 (sk) 1998-05-28 1999-05-28 Inhibítor VLA-4, farmaceutická kompozícia a ich použitie na prípravu liečiva
IL13996799A IL139967A (en) 1998-05-28 1999-05-28 Beta-alanine derivatives and pharmaceutical compositions containing the same
AT99926019T ATE256659T1 (de) 1998-05-28 1999-05-28 Ein vla-4-inhibitor: omepupa-v
MXPA00011774A MXPA00011774A (es) 1998-05-28 1999-05-28 Un nuevo inhibidor vla-4: ome pupa-v.
SI9930526T SI1082302T1 (en) 1998-05-28 1999-05-28 A vla-4 inhibitor: omepupa-v
EP99926019A EP1082302B1 (fr) 1998-05-28 1999-05-28 L'omepupa-v, nouvel inhibiteur du vla-4
PL344440A PL198189B1 (pl) 1998-05-28 1999-05-28 (R)-N-{[4-{[(2-metylofenyloamino)-karbonylo]amino}fenylo]acetylo}-L-prolilo-3-metylo]-ß-alanina, jej zastosowanie i kompozycja farmaceutyczna ją zawierająca
JP2000550827A JP2002516309A (ja) 1998-05-28 1999-05-28 新規ＶＬＡ−４阻害剤：ｏＭｅＰＵＰＡ−Ｖ
EA200001236A EA002988B1 (ru) 1998-05-28 1999-05-28 (r)-n-[[4-[[(2-метилфениламино)карбонил]амино]фенил]ацетил]-l-пропил-3- метил- бета-аланин и его применение в качестве ингибитора клеточной адгезии, опосредуемой интегрином альфа4бета1
CA002333656A CA2333656C (fr) 1998-05-28 1999-05-28 L'omepupa-v, nouvel inhibiteur du vla-4
NZ509199A NZ509199A (en) 1998-05-28 1999-05-28 A VLA-4 inhibitor: oMePUPA-V
HU0102255A HUP0102255A3 (en) 1998-05-28 1999-05-28 Vla-4 inhibitor omepupa-v, pharmaceutical compositions comprising thereof and their use
EEP200000698A EE04639B1 (et) 1998-05-28 1999-05-28 Raku adhesiooni pärssiv ühend ja seda sisaldav farmatseutiline kompositsioon
DK99926019T DK1082302T3 (da) 1998-05-28 1999-05-28 En VLA-4-inhibitor: oMePUPA-V
UA2000127603A UA65623C2 (en) 1998-05-28 1999-05-28 Vla-4: omepupa-v inhibitors
DE69913687T DE69913687T2 (de) 1998-05-28 1999-05-28 Ein VLA-4-Inhibitor: oMePUPA-V
IS5737A IS5737A (is) 1998-05-28 2000-11-28 Nýstárlegur VLA-4 tálmi: oMePUPA-V
US09/724,107 US6495525B1 (en) 1998-05-28 2000-11-28 VLA-4 inhibitor: oMePUPA-V
NO20006023A NO317990B1 (no) 1998-05-28 2000-11-28 Celleadhesjonsinhibitor samt farmasoytiske sammensetninger inneholdende denne
HK01106420A HK1035726A1 (en) 1998-05-28 2001-09-11 A vla-4 inhibitor: omepupa-v

Applications Claiming Priority (2)

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US8706498P 1998-05-28 1998-05-28
US60/087,064 1998-05-28

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US09/724,107 Continuation US6495525B1 (en) 1998-05-28 2000-11-28 VLA-4 inhibitor: oMePUPA-V

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WO1999061421A1 true WO1999061421A1 (fr) 1999-12-02

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US (1) US6495525B1 (fr)
EP (1) EP1082302B1 (fr)
JP (1) JP2002516309A (fr)
KR (1) KR100636713B1 (fr)
CN (1) CN1148350C (fr)
AT (1) ATE256659T1 (fr)
AU (1) AU764108B2 (fr)
BG (1) BG65021B1 (fr)
CA (1) CA2333656C (fr)
CZ (1) CZ298413B6 (fr)
DE (1) DE69913687T2 (fr)
DK (1) DK1082302T3 (fr)
EA (1) EA002988B1 (fr)
EE (1) EE04639B1 (fr)
ES (1) ES2211096T3 (fr)
HK (1) HK1035726A1 (fr)
HU (1) HUP0102255A3 (fr)
IL (1) IL139967A (fr)
IS (1) IS5737A (fr)
MX (1) MXPA00011774A (fr)
NO (1) NO317990B1 (fr)
NZ (1) NZ509199A (fr)
PL (1) PL198189B1 (fr)
PT (1) PT1082302E (fr)
SI (1) SI1082302T1 (fr)
SK (1) SK285280B6 (fr)
TR (1) TR200100190T2 (fr)
UA (1) UA65623C2 (fr)
WO (1) WO1999061421A1 (fr)
YU (1) YU75500A (fr)
ZA (1) ZA200007300B (fr)

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US6525069B1 (en) * 1998-12-18 2003-02-25 Bristol-Myers Squibb Pharma Co. N-ureidoalkyl-piperidines as modulators of chemokine receptor activity
US6630503B1 (en) 1999-08-13 2003-10-07 Biogen, Inc. Cell adhesion inhibitors
US6645939B1 (en) 1997-11-24 2003-11-11 Merck & Co., Inc. Substituted β-alanine derivatives as cell adhesion inhibitors
US6667331B2 (en) 1999-12-28 2003-12-23 Pfizer Inc Non-peptidyl inhibitors of VLA-4 dependent cell binding useful in treating inflammatory, autoimmune, and respiratory diseases
US7157487B2 (en) 2000-12-28 2007-01-02 Daiichi Pharmaceutical Co., Ltd. Vla-4 inhibitors
US7196112B2 (en) 2004-07-16 2007-03-27 Biogen Idec Ma Inc. Cell adhesion inhibitors
EP1814545A2 (fr) * 2004-11-09 2007-08-08 Irm, Llc Composes et compositions utilises comme inhibiteurs de la proteine kinase
US7618630B2 (en) 1998-09-14 2009-11-17 Board Of Regents, The University Of Texas System Methods of treating multiple myeloma and myeloma-induced bone resorption using integrin antagonists
US7691894B2 (en) 2003-07-24 2010-04-06 Daiichi Pharmaceutical Co., Ltd. Cyclohexanecarboxylic acid compound
US10335485B2 (en) 2010-04-16 2019-07-02 Biogen Ma Inc. Anti-VLA-4 antibodies
US11116760B2 (en) 2018-10-30 2021-09-14 Gilead Sciences, Inc. Quinoline derivatives
US11174256B2 (en) 2018-10-30 2021-11-16 Gilead Sciences, Inc. Imidazopyridine derivatives
US11179383B2 (en) 2018-10-30 2021-11-23 Gilead Sciences, Inc. Compounds for inhibition of α4β7 integrin
US11224600B2 (en) 2018-10-30 2022-01-18 Gilead Sciences, Inc. Compounds for inhibition of alpha 4 beta 7 integrin
US11578069B2 (en) 2019-08-14 2023-02-14 Gilead Sciences, Inc. Compounds for inhibition of α4 β7 integrin

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WO2008017025A2 (fr) * 2006-08-02 2008-02-07 Genzyme Corporation Thérapie combinée
AU2008219007A1 (en) 2007-02-20 2008-08-28 Merrimack Pharmaceuticals, Inc. Methods of treating multiple sclerosis by administration of alpha-fetoprotein in combination with an integrin antagonist
CA2721093A1 (fr) 2008-04-11 2009-10-15 Merrimack Pharmaceuticals, Inc. Lieurs d'albumine de serum humain, et ses conjugues
JP5677972B2 (ja) 2008-11-18 2015-02-25 メリマック ファーマシューティカルズ インコーポレーティッド ヒト血清アルブミンリンカーおよびそのコンジュゲート
WO2014036520A1 (fr) 2012-08-30 2014-03-06 Merrimack Pharmaceuticals, Inc. Polythérapies comprenant des agents anti-erbb3

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EP1034164A1 (fr) * 1997-11-24 2000-09-13 Merck & Co., Inc. Derives d'alanine-beta agissant en tant qu'inhibiteurs de l'adhesion cellulaire
EP1034164A4 (fr) * 1997-11-24 2001-07-11 Merck & Co Inc Derives d'alanine-beta agissant en tant qu'inhibiteurs de l'adhesion cellulaire
US6645939B1 (en) 1997-11-24 2003-11-11 Merck & Co., Inc. Substituted β-alanine derivatives as cell adhesion inhibitors
US7211252B2 (en) 1998-09-14 2007-05-01 Board Of Regents, The University Of Texas System Methods of treating multiple myeloma and myeloma-induced bone resorption using integrin antagonists
WO2000015247A3 (fr) * 1998-09-14 2000-05-25 Biogen Inc Traitement du myelome multiple et de la resorption osseuse provoquee par le myelome au moyen d'antagonistes de l'interaction -cam-1/vla-4
US7618630B2 (en) 1998-09-14 2009-11-17 Board Of Regents, The University Of Texas System Methods of treating multiple myeloma and myeloma-induced bone resorption using integrin antagonists
EP2065050A1 (fr) * 1998-09-14 2009-06-03 Board of Regents, The University of Texas System Procédés de traitement de myélome multiple et de résorption osseuse liée au myélome utilisant des antagonistes d'intégrine
WO2000015247A2 (fr) * 1998-09-14 2000-03-23 Board Of Regents, The University Of Texas System Traitement du myelome multiple et de la resorption osseuse provoquee par le myelome au moyen d'antagonistes de l'interaction -cam-1/vla-4
CZ302997B6 (cs) * 1998-09-14 2012-02-15 Board Of Regents, The University Of Texas System Použití protilátky nebo jejího fragmentu vázajícího antigen pro výrobu farmaceutického prípravku
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US7034043B2 (en) 1999-08-13 2006-04-25 Biogen Idec Ma Inc. Cell adhesion inhibitors
US6630503B1 (en) 1999-08-13 2003-10-07 Biogen, Inc. Cell adhesion inhibitors
US6903128B2 (en) 1999-12-28 2005-06-07 Pfizer Inc Non-peptidyl inhibitors of VLA-4 dependent cell binding useful in treating inflammatory, autoimmune, and respiratory diseases
US6668527B2 (en) 1999-12-28 2003-12-30 Pfizer Inc. Non-peptidyl inhibitors of VLA-4 dependent cell binding useful in treating inflammatory, autoimmune, and respiratory diseases
US6667331B2 (en) 1999-12-28 2003-12-23 Pfizer Inc Non-peptidyl inhibitors of VLA-4 dependent cell binding useful in treating inflammatory, autoimmune, and respiratory diseases
US7157487B2 (en) 2000-12-28 2007-01-02 Daiichi Pharmaceutical Co., Ltd. Vla-4 inhibitors
US7691894B2 (en) 2003-07-24 2010-04-06 Daiichi Pharmaceutical Co., Ltd. Cyclohexanecarboxylic acid compound
US7893279B2 (en) 2003-07-24 2011-02-22 Daiichi Pharmaceutical Co., Ltd. Cyclohexanecarboxylic acid compound
US7196112B2 (en) 2004-07-16 2007-03-27 Biogen Idec Ma Inc. Cell adhesion inhibitors
JP2008519762A (ja) * 2004-11-09 2008-06-12 アイアールエム・リミテッド・ライアビリティ・カンパニー タンパク質キナーゼ阻害剤としての化合物および組成物
EP1814545A4 (fr) * 2004-11-09 2009-06-10 Irm Llc Composes et compositions utilises comme inhibiteurs de la proteine kinase
US8101608B2 (en) 2004-11-09 2012-01-24 IRM LLC, a Delware Limited Corporation Compounds and compositions as protein kinase inhibitors
EP1814545A2 (fr) * 2004-11-09 2007-08-08 Irm, Llc Composes et compositions utilises comme inhibiteurs de la proteine kinase
JP4917041B2 (ja) * 2004-11-09 2012-04-18 アイアールエム・リミテッド・ライアビリティ・カンパニー タンパク質キナーゼ阻害剤としての化合物および組成物
US10335485B2 (en) 2010-04-16 2019-07-02 Biogen Ma Inc. Anti-VLA-4 antibodies
US11083791B2 (en) 2010-04-16 2021-08-10 Biogen Ma Inc. Anti-VLA-4 antibodies
US11571477B2 (en) 2010-04-16 2023-02-07 Biogen Ma Inc. Anti-VLA-4 antibodies
US11116760B2 (en) 2018-10-30 2021-09-14 Gilead Sciences, Inc. Quinoline derivatives
US11174256B2 (en) 2018-10-30 2021-11-16 Gilead Sciences, Inc. Imidazopyridine derivatives
US11179383B2 (en) 2018-10-30 2021-11-23 Gilead Sciences, Inc. Compounds for inhibition of α4β7 integrin
US11224600B2 (en) 2018-10-30 2022-01-18 Gilead Sciences, Inc. Compounds for inhibition of alpha 4 beta 7 integrin
US11578069B2 (en) 2019-08-14 2023-02-14 Gilead Sciences, Inc. Compounds for inhibition of α4 β7 integrin

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SK285280B6 (sk) 2006-10-05
CN1307561A (zh) 2001-08-08
ZA200007300B (en) 2002-02-27
IS5737A (is) 2000-11-28
ATE256659T1 (de) 2004-01-15
HUP0102255A3 (en) 2001-12-28
YU75500A (sh) 2002-12-10
EA002988B1 (ru) 2002-12-26
CA2333656A1 (fr) 1999-12-02
NO20006023L (no) 2001-01-17
UA65623C2 (en) 2004-04-15
SK18102000A3 (sk) 2001-07-10
NO20006023D0 (no) 2000-11-28
EE200000698A (et) 2002-06-17
DK1082302T3 (da) 2004-04-26
CZ20004425A3 (en) 2001-05-16
KR20010043906A (ko) 2001-05-25
HUP0102255A2 (hu) 2001-11-28
IL139967A0 (en) 2002-02-10
AU764108B2 (en) 2003-08-07
US6495525B1 (en) 2002-12-17
DE69913687T2 (de) 2004-10-07
JP2002516309A (ja) 2002-06-04
EE04639B1 (et) 2006-06-15
MXPA00011774A (es) 2002-10-17
PL198189B1 (pl) 2008-06-30
CZ298413B6 (cs) 2007-09-26
EP1082302A1 (fr) 2001-03-14
DE69913687D1 (de) 2004-01-29
HK1035726A1 (en) 2001-12-07
SI1082302T1 (en) 2004-06-30
AU4219299A (en) 1999-12-13
PT1082302E (pt) 2004-04-30
TR200100190T2 (tr) 2001-05-21
BG105060A (en) 2001-08-31
ES2211096T3 (es) 2004-07-01
CN1148350C (zh) 2004-05-05
NZ509199A (en) 2003-10-31
BG65021B1 (bg) 2006-12-29
KR100636713B1 (ko) 2006-10-20
IL139967A (en) 2005-11-20
EA200001236A1 (ru) 2001-06-25
NO317990B1 (no) 2005-01-17
EP1082302B1 (fr) 2003-12-17
CA2333656C (fr) 2008-07-29
PL344440A1 (en) 2001-11-05

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